This work involves the general characterization of proteins involved in bacterial respiration. The specific subjects of this proposal are the cytochrome claa3 and Rieske iron/sulfur protein isolated from the extremely thermophilic aerobe, Thermus thermophilus. The overall objective of the work is to provide physical insight on the mechanism of coupled proton translocation and electron transfer. The methods used include general techniques of protein purification and characterization; those of recombinant DNA technology to isolate and sequence the genes of interest; and physical methods including EPR, ENDOR, Mossbauer, and resonance Raman spectroscopies. Kinetic studies are designed for the detection and characterization of intermediates in dioxygen reduction, and for the detection of spectral perturbations of vesicular cytochrome aa3 under the influence of a polarizing electric field. Guided by the belief that understanding mechanisms in respiration will require detailed three-dimensional structures, we are making a serious effort to crystallize these proteins.